Angiotensin converting enzyme 2 (ACE2) activity in fetal calf serum: implications for cell culture research

Lubel, John S; Herath, Chandana B; Velkoska, Elena; Casley, David; Burrell, Louise M.; Angus, Peter W

doi:10.1007/s10616-009-9185-0

Cited by 7 publications

(3 citation statements)

References 27 publications

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“…Moreover, soluble ACE2 can effectively block the binding of SARS-CoV S-protein to membrane-bond ACE2 and thereby prevents SARS-CoV entry [ 13 , 14 ], which is possibly transferable to SARS-CoV-2. Since FCS contains soluble ACE2, a reduced FCS concentration possibly intensifies the infection of Caco-2 cells [ 15 ]. The cells were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01 for 24 h in the presence of the compounds.…”

Section: Methodsmentioning

confidence: 99%

Characterization of ACE Inhibitors and AT1R Antagonists with Regard to Their Effect on ACE2 Expression and Infection with SARS-CoV-2 Using a Caco-2 Cell Model

Reus

Schneider

Ulshöfer

et al. 2021

Life

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Blood-pressure-lowering drugs are proposed to foster SARS-CoV-2 infection by pharmacological upregulation of angiotensin-converting enzyme 2 (ACE2), the binding partner of the virus spike (S) protein, located on the surface of the host cells. Conversely, it is postulated that angiotensin–renin system antagonists may prevent lung damage caused by SARS-CoV-2 infection, by reducing angiotensin II levels, which can induce permeability of lung endothelial barrier via its interaction with the AT1 receptor (AT1R). Methods: We have investigated the influence of the ACE inhibitors (lisinopril, captopril) and the AT1 antagonists (telmisartan, olmesartan) on the level of ACE2 mRNA and protein expression as well as their influence on the cytopathic effect of SARS-CoV-2 and on the cell barrier integrity in a Caco-2 cell model. Results: The drugs revealed no effect on ACE2 mRNA and protein expression. ACE inhibitors and AT1R antagonist olmesartan did not influence the infection rate of SARS-CoV-2 and were unable to prevent the SARS-CoV-2-induced cell barrier disturbance. A concentration of 25 µg/mL telmisartan significantly reduced the virus replication rate. Conclusion: ACE inhibitors and AT1R antagonist showed neither beneficial nor detrimental effects on SARS-CoV-2-infection and cell barrier integrity in vitro at pharmacologically relevant concentrations.

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Section: Methodsmentioning

confidence: 99%

Characterization of ACE Inhibitors and AT1R Antagonists with Regard to Their Effect on ACE2 Expression and Infection with SARS-CoV-2 Using a Caco-2 Cell Model

Reus

Schneider

Ulshöfer

et al. 2021

Life

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“…Mouse proximal tubule (PT) cells derived from C57BL6 mice were grown in a defined medium of DMEM-F12 (1:1), supplemented with insulin (5 μg/ml), transferrin (5 μg/ml), selenium (5 ng/ml), hydrocortisone (50 nM), and 3,3′,5-triiodo-L-thyronine (2.5 nM), without fetal bovine serum (FBS). Serum has previously been demonstrated to contain ACE2 enzymatic activity, and should be avoided in this assay [15]. All biological samples are collected and placed on ice, aliquoted and then centrifuged at 12,000 × g for 5 min at 4 °C.…”

Section: Generation Of Standard Curves Using Human or Mouse Race2mentioning

confidence: 99%

Measurement of Angiotensin Converting Enzyme 2 Activity in Biological Fluid (ACE2)

Xiao

Burns

2017

Methods in Molecular Biology

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Angiotensin-converting enzyme 2 (ACE2) is a recently described member of the renin-angiotensin system that hydrolyzes angiotensin (Ang) II to Ang-(1-7), and may thereby protect against cardiovascular and renal diseases. ACE2 is a type 1 integral membrane protein and contains a catalytically active ectodomain that can be shed from the cell surface into the extracellular space, via cleavage by a disintegrin and metalloproteinase-17 (ADAM-17). ACE2 enzymatic activity and protein can be detected in biological fluids, including urine, plasma, and conditioned cell culture media. We present a detailed method for measurement of ACE2 activity in biological fluids, using hydrolysis of an intramolecularly quenched fluorogenic ACE2 substrate, in the absence or presence of the ACE2 inhibitors MLN-4760 or DX600. Recombinant human or mouse ACE2 is used to generate standard curves for this assay, with ACE2 detection ranging from 1.56 to 50 ng/ml. While MLN-4760 potently inhibits the activity of both human and mouse ACE2, DX600 (linear form) only effectively blocks human ACE2 activity in this assay. In biological samples of human and mouse urine, cell culture medium from mouse proximal tubular cells, and mouse plasma, the mean intra- and inter-assay coefficients of variation (CVs) of the assay range from 1.43 to 4.39 %, and from 7.01 to 13.17 %, respectively. We present data on the time and substrate concentration dependence of the assay, and show that exogenous D -glucose, creatinine, urea, and albumin do not interfere with its performance. In biological fluids, this assay is a simple and reliable method to study the role of ACE2 and its shed fragments in cardiovascular and renal diseases.

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“…Membrane ACE2 is detected through flow cytometry and represented as mean fluorescence intensity. The activity of soluble ACE2 is detected using the supernatant of 80% confluent cell lines after three days of culture as previously described 1 . B.…”

Section: Resultsmentioning

confidence: 99%

TMPRSS2 activation of Omicron lineage Spike glycoprotein is regulated by Collectrin-like domain of ACE2

Aggarwal,

Fichter,

Milogiannakis

et al. 2023

Preprint

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Continued high levels spread of SARS-CoV-2 globally enabled accumulation of changes within the Spike glycoprotein, leading to resistance to neutralising antibodies and concomitant changes to entry requirements that increased viral transmission fitness. Herein, we demonstrate a significant change in ACE2 and TMPRSS2 use by primary SARS-CoV-2 isolates that occurred upon arrival of Omicron lineages. Mechanistically we show this shift to be a function of two distinct ACE2 pools based on cleavage or non-cleavage of ACE2 by TMPRSS2 activity. In engineered cells overexpressing ACE2 and TMPRSS2, ACE2 was cleaved by TMPRSS2 and this led to either augmentation or progressive attenuation of pre-Omicron and Omicron lineages, respectfully. In contrast, TMPRSS2 resistant ACE2 restored infectivity across all Omicron lineages through enabling ACE2 binding that facilitated TMPRSS2 spike activation. Therefore, our data support the tropism shift of Omicron lineages to be a function of evolution towards the use of uncleaved pools of ACE2 with the latter consistent with its role as a chaperone for many tissue specific amino acid transport proteins.

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Angiotensin converting enzyme 2 (ACE2) activity in fetal calf serum: implications for cell culture research

Cited by 7 publications

References 27 publications

Characterization of ACE Inhibitors and AT1R Antagonists with Regard to Their Effect on ACE2 Expression and Infection with SARS-CoV-2 Using a Caco-2 Cell Model

Characterization of ACE Inhibitors and AT1R Antagonists with Regard to Their Effect on ACE2 Expression and Infection with SARS-CoV-2 Using a Caco-2 Cell Model

Measurement of Angiotensin Converting Enzyme 2 Activity in Biological Fluid (ACE2)

TMPRSS2 activation of Omicron lineage Spike glycoprotein is regulated by Collectrin-like domain of ACE2

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